1. Field of the Invention
The present invention relates to the field of cellular growth factor receptors, particularly to novel forms of epidermal growth factor receptor (EGFR), involving deletions, to molecular techniques for analyzing and producing the novel forms of EGFR, to other compositions relating to the novel proteins and genetic constructs encoding same, and to potential uses of the constructs.
2. Related Art
The epidermal growth factor receptor, or EGF receptor, is a transmembrane receptor protein that binds epidermal growth factor (EGF). Following the binding of EGF, the intrinsic tyrosine kinase activity of the receptor is activated, which leads to the phosphorylation of numerous substrates. The signaling cascade that is initiated has a significant role in driving cells to divide. The EGF receptor is also known to be involved in enhancing cell motility, invasiveness and survival among many other phenotypes. There is increased expression of the protein in numerous types of cancers, and overexpression has been shown to enhance the tumorigenic properties of cancer cells as well as to transform normal cells into cancer cells. For these reasons, the EGF receptor has been a target for several anti-cancer therapies. Two small molecule inhibitors of the tyrosine kinase activity of the receptor have received FDA approval and are now marketed as therapies for several cancers including non-small cell carcinoma of the lung. Antibodies directed against the EGF receptor have also been approved by the FDA as anti-cancer therapeutics.
On the other hand, stimulation of the EGF receptor can actually be beneficial in some cases. For example, in skin wounds the application of EGF can promote healing.
Several variant forms of the EGF receptor have been described. The most common alteration is known as EGF receptor variant III, or EGFRvIII. This represents the skipping of exons 2 through 7 resulting in the fusion of exon 1 to exon 8. The amino acid sequence represents the joining of amino acid 5 of the mature protein to amino acid 274 and the generation of a unique glycine at the junction. The protein alteration is characterized by the amino acid sequence LEEKKGNYVVTDH, (SEQ ID NO: 1) where the underlined G represents the glycine created at the fusion junction. The “mature form” lacks the first 25 amino acids found in the pro peptide and begins with a leucine, as can be seen in the sequences disclosed. Its full length would therefore not include the signal sequence, would include substantially all of the peptide to the carboxy terminus.
There are also mutations within the EGF receptor sequence that either alter single amino acids or delete small regions of the protein. These alterations have been found to enhance sensitivity of the EGF receptor to small molecule tyrosine kinase inhibitors and their presence can reasonably predict patient response to these drugs.
While the membrane localization of the EGF receptor appears critical to some cellular functions, other intracellular localizations of the receptor have been described that also have some role in cell physiology. The EGF receptor has been found in the Golgi and endoplasmic reticulum where it is thought that the signals generated by the receptor in these compartments are relevant to cell growth and perhaps oncogenesis. More interestingly, immunohistochemical studies with antibodies directed against the EGF receptor have consistently identified reactivity in the nucleus in some normal physiologic states but especially in human cancer tissues such as breast and thyroid cancer. However, the nuclear localization was only found using antibodies directed against the intracytoplasmic domain and was not seen with antibodies against the extracellular domain. In vitro studies have found that the intact EGF receptor can translocate to the nucleus, either using an intrinsic nuclear localization signal or a sec translocon accessory protein that mediates nuclear transport. This nuclearly localized receptor is capable of activating the transcription of several genes involved in cell growth and the promotion of the cell cycle. It has further been shown that the EGF receptor can either bind directly or indirectly to the promoter region of these transcription factors, thus directly participating in the initiation of transcription. Intriguingly, a synthetic molecule that represents the intracytoplasmic domain of the receptor has a higher efficiency of nuclear translocation and activation of transcription factors.
The human epidermal growth factor receptor (EGFR) is identified as NCBI Gene EGFR. The full length DNA sequence referenced here is found at X00588. Additional full length EGFR sequence information is also publicly known, e.g., NM_005228. EGFR isoforms a, b, c and d have been reported.